March 15, 2015 (Vol. 35, No. 6)

Where Promise Meets Reality

Disposability as a general biomanufacturing strategy arose from analysis of single-use economics, particularly with respect to cleaning and related validation for fixed-tank systems. Cost benefits were bolstered by the desire for versatility and agility, a general move from dedicated plants to multiproduct facilities, and the emergence of highly potent therapeutics that did not require huge bioreactors to fulfill worldwide demand.

Upstream single-use applications included media preparation and product hold, followed by cell culture. Single-use fermentation is not that far off. Downstream, filtration, and product hold are the most applicable areas for disposables. But for cost reasons chromatography remains a hold-out.

The key is balancing the cost of goods (COG) contribution of resins against column packing and priming, and resin regeneration. The success of prepacked columns demonstrates that bioprocessors are willing to pay more to eliminate the tribulations of that activity.

Bioprocessors have looked to alternative unit operations to replace various column separations. Single-use membrane adsorbers are now routine for some polishing steps. More distant are precipitation or crystallization to replace capture chromatography. Even further, perhaps, are inexpensive resin alternatives to Protein A, ion exchange, hydrophobic interaction, etc.

The final determinant in the feasibility of single-use chromatography, however, may be the biopharmaceutical market itself.

“Many niche or low-dose biopharmaceuticals don’t require very large bioreactors,” explains Michael Phillips, Ph.D., director of bioprocess development at EMD Millipore. “Those manufacturers tend to favor flexibility and are likely to manage multiproduct facilities. That is where single-use or single-campaign usage may make sense.”

Similarly, processes designed for clinical trial supply tend to be small-scale. It may not make sense, Dr. Phillips says, to recycle and store small quantities of resins after the required three or four batches are manufactured.

Mobius FlexReady from EMD Millipore is a single-use platform that can facilitate switching between chromatography and tangential flow filtration steps.

Opportunity in Biosimilars

Another potential market for throw-away resins is biosimilars, where for regulatory reasons producers desire flexibility and local production, and may therefore be averse to capital expenditures. They will still need to face the consequences of under-utilizing expensive chromatography media.

In today’s practice, resins do not contribute inordinately to COG precisely because they are recycled. But if used only a handful of times their contribution becomes significant. This conundrum is at least partly resolved by squeezing every bit of value from resins. This has been most successfully accomplished by multicolumn continuous chromatography.

Continuous chromatography often employs small, pre-packed columns that are cycled 30–50 times within a single run or up to 200 cycles overall,” Dr. Phillips says. “You’re able to fully utilize the resin lifetime within a few runs, thereby minimizing the impact to COG.

Fullest Resin Utilization

The take-home lesson from experts is that large-scale single-use chromatography will come about when resin contributions to COG approach those of other single-use products. One way to achieve this is through high binding capacities, but one can squeeze only so much protein into a column of a given volume. The other is to recycle, regenerate, and reuse resins continuously. That is the idea behind continuous chromatography systems.

One continuous approach causing a buzz these days is ChromaTan’s Continuous Countercurrent Tangential Chromatography™ (CCTC), which company president and founder Oleg Shinkazh calls “the future of column-free purification.”

Although it uses off-the-shelf resins CCTC does not employ a column in the traditional sense. Resin instead flows in a slurry through a series of mixers and hollow-fiber membranes where they sequentially bind product, are washed, eluted, and stripped to begin the process over again.

Resins undergo “countercurrent staging,” where the buffers used in the aforementioned steps flow against the resin slurry flow. According to Shinkazh this conserves buffer, as well as utilizes resin to its fullest. In studies published in leading separations and biotechnology journals, CCTC has achieved protein purities of greater than 99% with recoveries of 94%. Viewed from the perspective of process productivity, CCTC is capable of purifying 40–60 g of protein per liter of resin per hour, which is 5–10 times higher than for conventional packed columns.

“Columns can run up to three cycles per shift,” notes Shinkazh. “Our system runs four cycles per hour.”

Apropos to this discussion, CCTC is continuous, economic, column-free, scalable, and thanks to its resin utilization, single-use.

Because it is continuous CCTC does not produce peaks, but a flat output in which protein comes off at constant concentration. Contact time during elution is less than a minute, which is ideal for sensitive proteins. And the steady-state output allows seamless integration with subsequent process steps.

Shinkazh is modest about his accomplishment. “We’re not building a new device. We’re taking off-the-shelf products like hollow-fiber membranes and commercial resins, and putting them into a new format—a sophisticated tubing set. The buffer chemistries are similar, and we get the same purities and yields as column-based processes.”

Eventually, as the technology gains traction, Shinkazh will invest in designing resins and membranes specifically for CCTC.

ChromaTan has been going through speaking and publishing circuits to promulgate its message. The company’s roster of corporate and academic partners includes ASI (single-use systems), Fujifilm Diosynth Biotechnologies (contract manufacturing), Regeneron (pharmaceuticals), Spectrum Laboratories (bioseparations), and the chemical engineering department at Penn State.

CCTC uses approximately one-tenth the resin of a typical column to achieve equivalent results. “But costs are not one-tenth because resins do not comprise 100% of separation costs. But because of our high resin utilization, we are able to approach single use,” Shinkazh tells GEN.

The technology is not for sale yet as it’s still in beta testing, notes Shinkazh. “We are working with several groups to polish it, to get to the final design of a commercial system.”

ChromaTan has developed Continuous Countercurrent Tangential Chromatography (CCTC), which combines off-the-shelf components and a column-free approach. In this image, four black cassettes can be seen encasing the membrane/static mixer/tubing sets. The entire flow path is irradiated, and then installed in a modular manner. This includes the disposable pump heads (Watson Marlow) that snap into the pump drive.

Past and Future

Fredrik Lundström, product manager at GE Healthcare Life Sciences, points out that single-use chromatography was initially adopted for early product development, but now enters the scene at later development stages. “That said, there are scale limitations with single-use chromatography. Single-use will continue to be limited for very large scale manufacturing, where its benefit is smaller and where we may see hybrid solutions,” he explains.

The trend toward high-capacity, high-flow resins helps to reduce column sizes. There is also a trend toward smaller production batches and also the potential for continuous chromatography.

GE launched the first complete single-use chromatography system in 2008. Today, Lundström predicts “improved technology” that will enable larger scale.

“There is also now interest in continuous processing, which will bring down the scale of chromatography significantly in terms of both reduced column size and system capacity,” adds Lundström. “However, it will take some time before this is more broadly implemented.”

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